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Phosphatidylserine (PS) is a membrane phospholipid which in intact cells is exclusively localized in the inner leaflet of the lipid bilayer. However, once cells undergo apoptosis or oxidative stress, PS molecules are exposed on the external surface of the cells and this contributes to their adherence to macrophages or endothelial cells. PS exposure on Plasmodium falciparum-infected red cells was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS. Involvement of exposed PS in the adherence of malaria-infected red cells to endothelial cells was examined by in vitro cytoadherence assays. Infected cells exposed PS on their surface as the intracellular parasites matured to trophozoite and schizont stages. Adherence of malaria-infected cells to CD36, CD36-expressing Chinese hamster ovary cells, thrombospondin, and C32 amelanotic melanoma cells was inhibited by annexin V, whereas ICAM-1- and chondroitin sulfate A-mediated binding was not. Further, PS liposomes and glycerophosphorylserine, but not phosphatidylcholine liposomes and glycerophosphorylcholine, inhibited the binding of infected cells to CD36 and thrombospondin. In conclusion, these results demonstrate that PS exposed on the surface of malaria-infected red cells contributes, in part, to the adherence of P. falciparum-parasitized red cells to CD36 and thrombospondin.

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs x tLm -2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs x pm-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.

No abstract

Synthetic peptides patterned on the amino acid sequences found in two exofacial regions of band 3 protein (residues 824-829 of loop 7 and residues 547-553 of loop 3) blocked, in a dose-dependent fashion, the in vitro adherence of Plasmodium falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Intravenous infusion of these synthetic peptides into Aotus and Saimiri monkeys infected with sequestering isolates of P. fakciparum resulted in the appearance of mature forms of the parasite in the peripheral circulation. The finding that the peptides were effective as adhesion blockers in the micromolar range suggests that cerebral malaria could be managed through antiadhesion therapy.The hallmark ofPlasmodiumfalciparum infections is sequestration-that is, the attachment of erythrocytes infected with mature-stage parasites (trophozoites/schizonts) to the endothelial cells lining the postcapillary venules (1). In humans, the principal organs in which sequestration takes place are the heart, lung, kidney, and liver (2). Sequestration in the brain microvessels-a special pathology of P. falciparum infections called cerebral malaria-may occlude blood flow and result in confusion, lethargy, and deep coma (3). Although the plasmodial molecules on the surface of the malaria-infected erythrocyte that are responsible for binding to the endothelium have not been identified, a parasite-encoded protein, P. falciparum erythrocyte membrane protein 1 (PfEMP 1; ref. 4), similar to a recently described protein called sequestrin (5), has been correlated with cytoadherence. However, the precise role of PfEMP 1 (or sequestrin) in erythrocyte sequestration has not been determined.Earlier work (5-7) indicated that infection of erythrocytes by the malaria parasite P. falciparum leads to truncated forms ofband 3 protein in the erythrocyte membrane and that these were involved in the cytoadherent behavior of the infected erythrocyte. Further, several monoclonal antibodies directed against exofacial loops 3 and 7 of band 3 protein blocked cytoadherence (I.C. and I.W.S., unpublished results). In an attempt to further define the regions of band 3 protein responsible for the cytoadherent behavior of infected erythrocytes, peptides patterned on the surface-exposed domains of band 3 were synthesized, and their ability to inhibit the in vitro and in vivo adherence of P. falciparuminfected erythrocyte was determined.The following observations were used to determine which sequences in the exofacial loops (Fig. 1) might contain the adhesive region: (i) Cytoadherence is strongly influenced by pH (16,17), consistent with a protonated histidine residue being near to, or part of, the adhesin site. (ii) Cytoadherence is strongly inhibited by the iodination of surface proteins of a P. falciparum-infected erythrocyte (18), implying that a tyrosine residue is near to, or part of, the adhesin site. (iii) The adhesin is trypsin sensitive (18), suggesting that a trypsin site (a lysine or arginine) is close to, or part of, the adhesive region. Bec...