A Novel Role for Von Willebrand Factor in the Pathogenesis of Experimental Cerebral Malaria

Abstract

Cerebral malaria (CM) is a major cause of death in young children in sub-Saharan Africa. Although the pathogenesis of this condition remains poorly understood, Plasmodium falciparum infection results in adhesion of infected erythrocytes (IE) to microvascular endothelium, and acute endothelial cell (EC) activation. Recent studies have shown that plasma VWF:Ag and VWF propeptide levels are markedly elevated in children with severe falciparum malaria. Moreover elevated VWF:Ag levels inversely correlate with clinical outcomes. In addition, circulating ultra-large VWF multimers have also been observed in children with severe malaria. Importantly, platelet-decorated UL-VWF strings have been shown to recruit trophozoite-stage falciparum-IE to EC surfaces in shear-based assays. Collectively, these emerging data suggest that VWF may play a novel role in the pathogenesis of CM. To further investigate this hypothesis, we utilized an established murine model of experimental CM which involves wild type (WT) C57BL/6J mice infected with Plasmodium berghei ANKA. In brief, WT C57Bl/6J mice were inoculated with 2x106P. berghei ANKA via I.P. injection. These mice typically developed CM and died within 6-8 days. In keeping with findings in children with falciparum malaria, acute EC activation was also an early and consistent feature in the murine model of CM. Mean plasma VWF:Ag levels were significantly elevated from Day+3 in infected mice compared to controls (Day +3 1.8 fold increase; Day +5 2.5 fold increase; p<0.01). In addition, plasma angiopoietin-2 and osteoprotegerin levels were also both significantly elevated from Day +3 and Day +5 respectively in mice infected with P. berghei. Interestingly, despite the fact that murine plasma ADAMTS13 activity levels were not significantly reduced, pathological ultra-large VWF multimers (UL-VWF) were also observed in murine plasma from Day +3 following P. berghei inoculation. Cumulatively these findings suggest that early marked elevation in VWF:Ag levels, and the appearance of pathological UL-VWF multimers in the plasma, represent hallmarks of both human and murine severe malaria infection. To determine whether VWF plays a direct role in modulating the pathogenesis of CM in vivo, we further investigated P. berghei infection in VWF-/- C57BL/6J mice. Importantly, although there was no difference in blood parasitaemia levels, overall survival was significantly prolonged in VWF-/- mice compared to wild type mice (6 versus 7.25 days; p=0.0106). Moreover, a significant delay in malaria clinical progression in the VWF-/- mice was also observed using a previously validated clinical scoring algorithm for experimental cerebral malaria. (Amante et al, Am J Path 2007). Recent studies have demonstrated that platelets may play a direct role in modulating malaria parasite killing. Consequently, to investigate potential mechanisms through which VWF-/- mice are protected against experimental CM, daily platelet counts were determined in WT and VWF-/- mice following infection. In keeping with observations in human patients infected with falciparum malaria, significant thrombocytopenia was also a consistent feature in the murine model. For example, by Day +4 following inoculation of WT mice mean platelet count had fallen by 71.6 ± 26 %. Interestingly however, significant thrombocytopenia was also observed in VWF-/- mice infected with P. berghei (84.2 ± 9 %). Despite the significant differences in clinical progression and overall survival no significant differences in platelet counts were observed between VWF-/- and WT mice at any time point. These findings suggest that VWF-/-mice are protected against CM through a platelet-independent mechanism. In conclusion, we demonstrate that early and consistent EC activation is a feature of the murine model of experimental CM similar to previous findings of children infected with P. falciparum. In addition, our findings show that marked elevation of VWF:Ag, and the appearance of pathological UL-VWF multimers in the plasma, both represent hallmarks of human and murine severe malaria infection. Finally, we report that VWF-/- mice exhibit significantly prolonged survival against CM, and that this effect is mediated through a platelet-independent mechanism. Given the significant morbidity and mortality associated with CM, these novel data have direct translational significance. Disclosures No relevant conflicts of interest to declare.

ADAMTS13 regulation of VWF multimer distribution in severe COVID‐19

“…Plasma VWF:Ag and VWF collagen binding activity (VWF:CB) were determined by ELISA and VWF sialylation assessed using modified lectin‐binding arrays as before 13 . VWF multimer analysis was performed as previously described 7 . ADAMTS‐13 activity was quantified using a commercial FRETS‐VWF73 assay (Peptides International, Inc.).…”

“…In normal plasma, VWF multimer distribution is regulated by the metalloprotease ADAMTS‐13 (A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13). Dysregulation of VWF proteolysis by ADAMTS‐13 is important in the etiology of a number of microangiopathies that share similar features to severe COVID‐19 (notably TTP and cerebral malaria) 7 . Although several studies have reported elevated plasma VWF levels in severe COVID‐19, 6,8–12 the data on VWF multimer patterns is less clear.…”

Contribution of a European‐Prevalent Variant near CD83 and an East Asian–Prevalent Variant near IL17RB to Herpes Zoster Risk in Tofacitinib Treatment: Results of Genome‐Wide Association Study Meta‐Analyses

“…The VWF gene encodes the protein von Willebrand factor (vWF), which functions as both an antihemophilic factor carrier and a platelet-vessel wall mediator in the blood coagulation system. The levels of vWF rise in multiple types of infections (38,39). In a candidate gene study, VWF genetic variants were associated with human herpes simplex encephalitis, a rare complication following infection with herpes simplex virus type 1, which usually remains latent in neurons (40).…”

Illustrated State‐of‐the‐Art Capsules of the ISTH 2021 Congress

“…Higher VWF levels correlate with worse clinical outcomes. Pathological ultra‐large (UL‐) VWF multimers are also a feature of cerebral malaria 47 . In vitro studies have demonstrated that UL‐VWF strings on the surface of activated EC can recruit platelets.…”

“…Pathological ultra-large (UL-) VWF multimers are also a feature of cerebral malaria. 47 In vitro studies have demonstrated that UL-VWF strings on the surface of activated EC can recruit platelets. Subsequently, the platelet-decorated UL-VWF strings can then tether malaria-infected red blood cells (IRBC) under physiological shear stress.…”