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The O‐linked glycans of human von Willebrand factor modulate its interaction with ADAMTS ‐13
Author(s) -
Nowak A. A.,
McKin T. A. J.,
Hughes J. M.,
Chion A. C. K.,
Laffan M. A.
Publication year - 2014
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12451
Subject(s) - von willebrand factor , adamts , chemistry , proteolysis , adamts13 , platelet , glycan , microbiology and biotechnology , linker , thrombospondin , biochemistry , metalloproteinase , immunology , biology , glycoprotein , matrix metalloproteinase , enzyme , computer science , operating system
Summary Background O‐linked glycans ( OLG s) are clustered on either side of the von Willebrand factor ( VWF ) A1 domain and modulate its interaction with platelets; however, their influence on the VWF interaction with ADAMTS ‐13 is unknown. Objectives To assess the role of the OLG s in VWF susceptibility to ADAMTS ‐13 proteolysis, which would help to explain their specific distribution. Methods OLG sites were mutated individually and as clusters on either and both sides of the A1 domain, and expressed in HEK 293T cells. First, their proteolysis by ADAMTS ‐13 was assayed in the presence of urea. Next, a parallel‐flow chamber was used to analyze VWF ‐mediated platelet capture on collagen in the presence and absence of ADAMTS ‐13 under a shear stress of 1500 s −1 . The decrease in platelet capture in the presence ADAMTS ‐13 was used as a measure of VWF proteolysis. Results Initially, we found that, under denaturing conditions, the C‐terminal S1486A and Cluster 2 and double cluster ( DC ) variants were less susceptible to ADAMTS ‐13 proteolysis than wild‐type VWF . Next, we showed that addition of ADAMTS ‐13 diminished VWF ‐mediated platelet capture on collagen under flow; surprisingly, this was more pronounced with the S1486A, Cluster 2 and DC variants than with wild‐type VWF , indicating that these are proteolyzed more rapidly under shear flow. Conclusions OLG s provide rigidity to peptide backbones, and our findings suggest that OLG in the A1–A2 linker region regulates VWF conformational changes under shear. Importantly, the impact of OLG s on ADAMTS ‐13 cleavage under shear stress is the opposite of that under denaturing conditions, highlighting the non‐physiologic nature of in vitro cleavage assays.