Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• ADAMTS13 is a metalloprotease crucial for regulating von Willebrand factor (VWF) activity.
• VWF multimer cleavage by ADAMTS13 prevents thrombotic microangiopathies.
• The specificity of ADAMTS13 for its substrate VWF is not fully understood.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying ADAMTS13's specificity for VWF.
• To identify the specific domains and residues involved in ADAMTS13-VWF interaction.
• To quantify the contribution of different interactions to substrate cleavage efficiency.

Main Methods:

• Kinetic analysis of recombinant ADAMTS13 constructs.
• Site-directed mutagenesis and internal deletions within VWF domain A2.
• Analysis of substrate cleavage rates.

Main Results:

• ADAMTS13 spacer domain binds to the VWF A2 C-terminal alpha-helix, contributing to specificity.
• Interactions between ADAMTS13 (TSP1, Cys-rich, spacer domains) and VWF A2 (Gln(1624)-Arg(1668)) increase cleavage rate ~300-fold.
• Cleavage requires interactions with residues flanking the Tyr(1605)-Met(1606) scissile bond, indicating metalloprotease domain involvement.

Conclusions:

• ADAMTS13 recognizes VWF through cooperative, modular contacts involving multiple ADAMTS13 domains and discrete VWF A2 segments.
• The distance between the scissile bond and auxiliary binding sites is not critical.
• Specific VWF recognition is mediated by a combination of exosite and active site interactions.