ADAMTS-13 glycans and conformation-dependent activity.

Essentials The impact of N-linked glycosylation on ADAMTS-13 function has not been fully explored. The activity of glycan modified ADAMTS-13 was investigated under static and shear stress conditions. Terminal sialic acid on the metalloprotease domain glycans are important for ADAMTS-13 activity. The CUB domain glycans modulate ADAMTS-13 activity. SUMMARY: Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

The O-linked glycans of human von Willebrand factor modulate its interaction with ADAMTS-13.

BACKGROUND: O-linked glycans (OLGs) 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 OLGs 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 HEK293T 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: OLGs 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 OLGs 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.

The von Willebrand factor predicted unpaired cysteines are essential for secretion.

BACKGROUND: von Willebrand factor (VWF) contains free thiols that mass spectroscopy has located to nine cysteines: two in the D3 domain (Cys889 and Cys898) and seven in the C domains (Cys2448, Cys2451, Cys2453, Cys2490, Cys2491, Cys2528, and Cys2533) (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). It has been suggested that these free thiols function to regulate the self-association of VWF through thiol-disulfide exchange (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). However, recent structural modeling has predicted that these cysteines are, in fact, disulfide-bonded (Blood, 118, 5312; Blood, 120, 449). OBJECTIVES: To use mutation and expression analyses to investigate how these conflicting reports might be compatible with the synthesis and expression of VWF. METHODS AND RESULTS: Both full-length VWF and VWF fragments with cysteine to alanine mutations of the nine cysteines and two predicted binding partners (Cys2431 and Cys2468) failed to secrete. Mutation of a cysteine pair, C2431A/C2453A, similarly resulted in a failure to secrete, indicating that this is not secondary to creation of an unpaired thiol. Deletion mutants containing seven of these cysteines, conforming to hypothesized domain boundaries, also failed to secrete: ∆C1C6 (2255-2720), ∆C3C4 (2429-2577), ∆C3 (2429-2496), and ∆C4 (2497-2577). Analysis of cell lysates and immunofluorescence confirmed that the mutants were retained within the endoplasmic reticulum (ER). Coexpression with wild-type VWF rescued secretion of some mutants to a limited extent. CONCLUSIONS: These data suggest: first, that pairing of cysteines implicated in free thiol exchange is essential for correct folding of the VWF molecule, and unpairing must occur following exit from the ER or secretion from the cell; and second, that intact C domains are essential for efficient VWF secretion and must interact in the ER.

Characterisation of von Willebrand factor A1 domain mutants I1416N and I1416T: correlation of clinical phenotype with flow-based platelet adhesion.

BACKGROUND: Type 2M von Willebrand disease (VWD) results from mutations in the A1 domain of von Willebrand factor (VWF) that reduce its platelet-binding function. However, currently employed VWF functional static assays may not distinguish between clinical phenotype. METHODS: Fifteen individuals from five kindreds with VWF-A1 domain mutations I1416T or I1416N, correlated with mild and moderate clinical phenotypes, respectively, were investigated. The mutations were reproduced by site-directed mutagenesis and expressed in HEK293T cells; functional studies of the recombinant mutants, including GPIbα binding using a flow-based assay, were performed. RESULTS: Plasma from all individuals demonstrated discordant reductions in VWF antigen and platelet-binding function in the presence of high-molecular-weight VWF multimers consistent with VWD type 2M. There was lowered expression and secretion of both mutants compared with wild type (WT) recombinant (r)VWF as well as a significant reduction in GPIbα binding. Binding to collagen was normal and electrophoretic analysis demonstrated a similar multimer distribution between the mutant proteins and wt-rVWF. GPIbα binding under flow was also significantly reduced for I1416N and I1416T rVWF. Impairment of GPIbα binding was more marked for I1416N rVWF than I1416T under both static and flow conditions: this was in spite of similar VWF:Ristocetin cofactor (RCo) activities in patient plasma and is consistent with a respective clinical phenotype. CONCLUSIONS: Our findings have established for the first time that I1416N and I1416T are responsible for a type 2M VWD phenotype and demonstrate that quantification of VWF function under shear stress may provide a more accurate measure of clinical severity than the static functional measurements in current diagnostic use.