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Published on: June 2, 2023
Zinc and calcium ions cooperatively modulate ADAMTS13 activity
Patricia J Anderson1, Koichi Kokame, J Evan Sadler
1Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Abstract:
ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced approximately 2-fold by zinc ions, approximately 3-fold by calcium ions, and approximately 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to approximately 200-fold by zinc ions (K(D) (app) approximately 0.5 microM), calcium ions (K(D) (app) approximately 4.8 microM), and barium ions (K(D) (app) approximately 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K(m) (app) of 3.7 +/- 1.4 microg/ml (approximately 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 microg/ml. ADAMTS13 could cleave approximately 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K(m) (app) of 3.2 +/- 1.1 microM, consistent with an approximately 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 microM(-1) min(-1) and 18 microM(-1) min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.
Insights
The metalloproteinase ADAMTS13 activity, crucial for cleaving von Willebrand factor (VWF), is significantly enhanced by zinc and calcium ions. This metal ion dependence highlights key factors influencing VWF multimer processing in plasma.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- ADAMTS13 is a key metalloproteinase responsible for cleaving von Willebrand factor (VWF) multimers.
- Understanding the regulation of ADAMTS13 activity is crucial for VWF-mediated platelet aggregation and thrombosis.
- Metal ion cofactors are known to influence the activity of many metalloproteinases.
Purpose of the Study:
- To investigate the metal ion dependence of ADAMTS13 activity.
- To characterize the activation of ADAMTS13 by zinc, calcium, and barium ions using both multimeric VWF and a peptide substrate.
- To determine the kinetic parameters of ADAMTS13 cleavage for VWF and a peptide substrate.
Main Methods:
- Assessed ADAMTS13 activity using multimeric VWF and a fluorescent peptide substrate (FRETS-VWF73).
- Examined the effect of various metal ions (Zn2+, Ca2+, Ba2+) on ADAMTS13 activity in plasma and purified systems.
- Determined kinetic parameters (Km, catalytic efficiency) for VWF and FRETS-VWF73 cleavage.
Main Results:
- ADAMTS13 activity was significantly enhanced by zinc (approx. 2-fold in plasma, up to 200-fold with recombinant enzyme) and calcium ions (approx. 3-fold in plasma, up to 200-fold with recombinant enzyme).
- Cooperative activation was observed with both zinc and calcium ions.
- Barium ions stimulated activity in citrated plasma, likely by chelating citrate and releasing bound Ca2+ and Zn2+.
- ADAMTS13 demonstrated a strong preference for VWF over the peptide substrate, suggesting specific structural recognition sites on VWF.
Conclusions:
- Zinc and calcium ions are potent activators of ADAMTS13, with synergistic effects observed.
- The activity of ADAMTS13 is modulated by the presence of chelating agents like citrate in plasma.
- ADAMTS13 exhibits substrate specificity, preferentially cleaving VWF multimers over smaller peptide fragments, indicating recognition of structural features on VWF.
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