Von Willebrand Factor (VWF) is a large multi-domain plasma glycoprotein that is
critical for normal platelet tethering during haemostasis. ADAMTS13 is a plasma
metalloprotease that regulates VWF multimeric size/function by cleaving the
Tyr1605-Met1606 bond in the VWF A2 domain. Deficiency of ADAMTS13 causes
microvascular thrombosis. The only reported cleavage of VWF by ADAMTS13 is
that of the Tyr1605-Met1606 bond. How this high substrate specificity is conferred
remains unclear. To date, all the interactions between these molecules that have been
described involve VWF residues C-terminal to the scissile bond and the non-catalytic
domains of ADAMTS13. I hypothesised that VWF also contains an interaction site
for ADAMTS13 N-terminal of the cleavage site to aid both in positioning of the
scissile bond in the active site and substrate specificity. Previous studies have
suggested that the VWF sequence between Asp1596 and Val1604, N-terminal to the
cleavage site, is essential for cleavage by ADAMTS13. My aim was to identify the
residues in this region that are important determinants for ADAMTS13 proteolysis. A
panel of mutations were introduced into the substrate VWF 115 (VWF residues 1554-
1668). The mutants were expressed purified and their proteolysis by ADAMTS13
analysed. It was found that the proteolysis of VWF 115 variants (L1603A, L1603S,
L1603N or L1603K) were all substantially impaired (up to >400 fold reduction). The
importance of VWF Leu1603 was confirmed using a synthetic
peptide 1596DREQAPNLVY1605, which competitively inhibited proteolysis of VWF
115 by ADAMTS13. A mutant peptide containing the L1603A
mutation, 1596DREQAPNAVY1605, had minimal effect. When the VWF L1603A
substitution was introduced into the full-length recombinant VWF, proteolysis by
ADAMTS13 was again substantially reduced. These findings implied the presence of
a subsite (S3) in the ADAMTS13 metalloprotease (MP) domain that interacts with
VWF Leu1603. Using molecular modelling, the distance between VWF Leu1603 and
the scissile bond was estimated as ~10Å. Structural homology modelling of the MP
domain, mutagenesis of 11 candidate residues and functional characterisation of these
variants identified two clusters, Leu198/Leu232/Leu274 and Val195/Leu151, as
possible subsites interacting with VWF. It is suggested that VWF Leu1603 interacts
with Leu198/Leu232/Leu274, while Val195/Leu151 may interact with VWF Tyr1605.
I propose a mechanism for VWF cleavage involving remote C-terminal domain
interactions that assist initial orientation of the VWF scissile bond within the active
site of ADAMTS13, but in which N-terminal hydrophobic interactions between VWF
Leu1603 and the S3 subsite in the MP domain of ADAMTS13 are critical for the
positioning required for cleavage of the Tyr1605-Met1606 scissile bond.