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 ...

We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13. (Blood.

Key Points• ECM is associated with an early marked increase in plasma VWF levels and accumulation of UL-VWF multimers.• Following P berghei infection, VWF 2/2 mice survive significantly longer compared with WT controls.Plasmodium falciparum malaria infection is associated with an early marked increase in plasma von Willebrand factor (VWF) levels, together with a pathological accumulation of hyperreactive ultra-large VWF (UL-VWF) multimers. Given the established critical role of platelets in malaria pathogenesis, these increases in plasma VWF raise the intriguing possibility that VWF may play a direct role in modulating malaria pathogenesis. To address this hypothesis, we used an established murine model of experimental cerebral malaria (ECM), in which wild-type (WT) C57BL/6J mice were infected with Plasmodium berghei ANKA. In keeping with findings in children with P falciparum malaria, acute endothelial cell activation was an early and consistent feature in the murine model of cerebral malaria (CM), resulting in significantly increased plasma VWF levels. Despite the fact that murine plasma ADAMTS13 levels were not significantly reduced, pathological UL-VWF multimers were also observed in murine plasma following P berghei infection. To determine whether VWF plays a role in modulating the pathogenesis of CM in vivo, we further investigated P berghei infection in VWF 2/2 C57BL/6J mice. Clinical ECM progression was delayed, and overall survival was significantly prolonged in VWF 2/2 mice compared with WT controls. Despite this protection against ECM, no significant differences in platelet counts or blood parasitemia levels were observed between VWF 2/2 and WT mice. Interestingly, however, the degree of ECMassociated enhanced blood-brain barrier permeability was significantly attenuated in VWF 2/2 mice compared with WT controls.Given the significant morbidity and mortality associated with CM, these novel data may have direct translational significance. (Blood. 2016;127(9):1192-1201) IntroductionPlasmodium falciparum malaria remains a major cause of morbidity and mortality among children in sub-Saharan Africa. [1][2][3] Although the biological mechanisms involved in the pathophysiology of severe P falciparum malaria remain poorly understood, previous studies have demonstrated that sequestration of P falciparum-infected erythrocytes (IEs) within the microvasculature of the brain is important in the development of cerebral malaria (CM). 4,5 This sequestration involves adhesion of IE to host vascular endothelial cell (EC) surfaces [6][7][8] and is mediated by a variety of specific EC adhesion molecules including CD36, intercellular adhesion molecule-1, and thrombospondin. 9 Moreover, recent studies have also demonstrated that the endothelial protein C receptor also plays an important role in modulating the sequestration of IE. 10 In addition to IE, sequestration of leukocytes and platelets within the cerebral microvasculature has also been reported in postmortem samples from CM patients. 11,12Von Willebrand factor (VWF)...