We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important. IntroductionVon Willebrand factor (VWF) is a large multimeric plasma glycoprotein essential to normal hemostasis, first acting as the carrier molecule for procoagulant factor VIII (FVIII), extending its half-life within the circulation by protecting it from proteolytic degradation, and second, supporting platelet adhesion to thrombogenic surfaces at sites of vascular injury. 1,2 Synthesis of VWF is limited to megakaryocytes and endothelial cells. 3 The pre-pro-VWF molecule comprises a 22-amino acid signal peptide, a 741-amino acid propeptide, and the 2050-amino acid mature subunit. The pro-VWF monomer is composed of 4 types of domains (A-D) arranged as follows: NH 2 -D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, with the largest multimers exceeding 2 ϫ 10 4 kDa and being the most hemostatically active. Within the circulation, the multimeric size of VWF is controlled by the plasma metalloprotease ADAMTS13, which cleaves VWF at the Y1605-M1606 bond within the A2 domain, reducing multimeric size and thus regulating its adhesive function. 4 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer. 5 The overall structural composition of the glycans has been determined, but their exact functional significance is poorly understood. There is some evidence to suggest they protect the molecule from proteolytic degradation and are required for dimerization and subsequent multimerization. [6][7][8] Significantly, a small proportion of the N-linked glycans on VWF present the ABO(H) blood group sugars, 9,10 and the importance of this is highlighted by the well-established ...