Abstract Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM). This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.