Heparan sulfate dependent sequestration during Plasmodium falciparum malaria

Abstract: Plasmodium falciparum infected erythrocytes are able to bind to the microvascular endothelium (cytoadherence), and to uninfected erythrocytes (rosetting) that lead to excessive sequestration of the parasites. This causes obstruction of the deep microvasculature by P. falciparum infected erythrocytes (parasitized red blood cell (pRBCs)), which is partly believed to be responsible for the severe symptoms that evolve during malaria. Glycan receptors expressed on target cells, which are involved in adhesion of pRBCs, have been studied and heparan sulfate (HS) was identified as a comprehensive receptor implicated both in cytoadherence and in rosetting. Enzymatic elimination of HS on endothelial cells reduced the binding of pRBCs more then 70% and in competitive assays with soluble HS the adhesion was completely inhibited. The parasite derived protein Plasmodium falciparum erythrocyte membrane protein I (PfEMP1) is believed to be the major surface antigen involved in sequestration. By analysing individual domains of PfEMP1, the N-terminal Duffy binding like domain-1alpha (DBL1alpha) was shown to be the ligand binding to 14S on the endothelial cells. The analogous receptor on human red blood cells (RBCs), which is involved in rosetting and binds to DBL1alpha, has previously been described as a HS-like GAG. Here it was shown that HS is also present on mature human RBCs. This was demonstrated by the isolation of large negatively charged 0-glycans that were sensitive to HS specific reactions. Purified glycans from RBCs also showed affinity for DBL1alpha indeed suggesting HS to be a receptor involved in rosetting. In addition, by analysing biotinylated human RBC membranes a core protein from a HS-proteoglycan of about 30 kDa was identified. To study in vivo sequestration of P. falciparum infected human RBCs and the induced pathology, a small animal model was developed using Sprague-Dawley rats and enriched 99mTechnetium labelled pRBCs. Injection of pRBCs showed measurable sequestration in the rat lungs as compared with injection of solely RBCs. The sequestration of pRBCs was found to be dependent on surface ligands as digestion of adhesive proteins on the pRBC surface with trypsin reduced sequestration. Different parasite strains and clones showed distinct levels of sequestrated material and from histological examinations the different parasites seemed to prefer adhesion in the smaller or the larger microvasculature and generate diverse intravascular changes. The rat model was further used to evaluate the anti- sequestration effects of the HS analogous glycan heparin that was chemically modified to inactivate the anti-coagulant activity (inactivated heparin (IA-heparin)). In vitro studies showed that the IA-heparin bound to the pRBC surface and to recombinant DBL1alpha, disrupted rosettes and reduces cytoadherence. In vivo, the IA-heparin blocked sequestration of pRBCs in rat lungs up to 80%. Taken together, the results presented in this thesis will hopefully improve the knowledge of the molecular mechanisms underlying severe malaria and allow the development of a prototypic antisequestration drug in order to alleviate the symptoms of severe malaria.