N-linked glycans at N1515 and N1574 are critical determinants of VWF clearance in vivo. (A) A model of the VWF A2 domain was prepared as previously described.⁶⁶  Mass spectrometry analysis of human pd-VWF has provided extensive information regarding the N-glycome of VWF. Utilizing this information, a model of the VWF A2 domain with its associated glycans was constructed using Glycam Glycoprotein Builder software. N1515 and N1574 glycans structures were mapped onto the A2 domain crystal structure using this glycan modeling. This in silico analysis revealed that the complex glycans at N1515 and N1574 were both of significant size, spanning ∼33 Å and ∼36 Å in length, respectively. (B) To investigate a potential role for specific glycan sites in influencing VWF clearance, N1515 and N1574 in the A2 domain were targeted for removal by site-directed mutagenesis (VWF-N1515Q and VWF-N1574Q, respectively). In vivo clearance studies of these VWF glycan variants were performed as before and compared with WT rVWF. (C) Given that the glycans N1515 and N1574 reside within the A2 domain of VWF, we further sought to examine if these glycans could also influence the in vivo survival of an A1A2A3 VWF truncated fragment. To this end, site-directed mutagenesis was performed to eliminate the glycan at N1515 (A1A2A3-N1515Q) and N1574 (A1A2A3-N1574Q). Clearance examined in VWF^−/− mice as before. All results are plotted as percentage residual VWF:antigen levels relative to the amount injected. Data are presented as mean ± SEM.