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)...

Key Points• The A1 domain of VWF contains a cryptic binding site that plays a key role in regulating macrophage binding and clearance.• The N-linked glycans presented at N1515 and N1574 within the A2 domain of VWF modulate macrophage-mediated clearance.Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3.Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo. (Blood. 2016;128(15):1959-1968

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of - vs-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF -glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal -acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.