47 Am. J. Trop. Med. Hyg., 65(1), 2001, pp. 47–51 Copyright  2001 by The American Society of Tropical Medicine and Hygiene BINDING OF PLASMODIUM FALCIPARUM-INFECTED ERYTHROCYTES TO SOLUBLE PLATELET ENDOTHELIAL CELL ADHESION MOLECULE-1 (PECAM-1/CD31): FREQUENT RECOGNITION BY CLINICAL ISOLATES ANDREAS HEDDINI, QIJUN CHEN, JACK OBIERO, OSCAR KAI, VICTOR FERNANDEZ, KEVIN MARSH, WILLIAM A. MULLER, AND MATS WAHLGREN Microbiology and Tumor Biology Center, Karolinska Institutet and Swedish Institute for Infectious Disease Control, Stockholm, Sweden; Kenya Medical Research Institute, Kilifi Unit, Kilifi, Kenya; Department of Pathology, Weill Medical College of Cornell University, New York, New York Abstract. Platelet-endothelial cell adhesion molecule-1 or CD31 (PECAM-1/CD31) is a receptor recognized by Plasmodium falciparum–parasitized erythrocytes (pRBCs). Fluorescence-labeled soluble recombinant PECAM-1/ CD31 (sPECAM-1/CD31) is shown to bind to the surface of P. falciparum-infected erythrocytes on up to 70% of the cells. Binding is blocked by the addition of the unlabeled receptor in a dose-dependent fashion, but not by unrelated receptor-proteins. A significant correlation was found between the binding of sPECAM-1/CD31 to pRBCs and the binding to transfected L cells expressing the receptor as seen with six different P. falciparum lines or clones. Panning of cultures on PECAM-1/CD31 transfected L cells was paralleled by an increase in the binding of sPECAM-1/CD31. The pRBCs of 54% of fresh patient-isolates bound sPECAM-1/CD31 with a mean rate of 12.9% (range = 1.1–44%). The data suggest that PECAM-1/CD31 is a common receptor recognized by wild isolates and that the soluble PECAM- 1/CD31 suspension assay is a sensitive and reliable way to study PECAM-1/CD31 binding. INTRODUCTION Malaria infection caused by Plasmodium falciparum has two unique features that separate it from other forms of hu- man malaria: sequestration of parasitized red blood cells (pRBCs) in the microvasculature and the occurrence of se- vere pathologic complications. 1 It is generally believed that the two phenomena are linked and that massive adhesion of pRBCs to the vascular endothelium may lead to occlusion of the microvasculature and thereby play an important role in the pathogenesis of cerebral malaria. 2,3 Furthermore, the inflammatory response with the production of cytokines, such as tumor necrosis factor- 4,5 and nitric oxide, 6 has been suggested to participate in the pathogenesis of the disease. Examining laboratory-selected parasites in cell cytoadher- ence assays has identified a number of endothelial cell re- ceptors. These include the adhesion molecules CD36, 7 plate- let-endothelial cell adhesion molecule-1 (PECAM-1/CD31), 8 intercellular adhesion molecule-1, (ICAM-1), 9 thrombospon- din (TSP), 10 and glycosaminoglycan chondroitin sulfate A (CSA). 11 Ex vivo, CD36 is the most frequent target of strains from patients with mild as well as severe P. falciparum ma- laria, but it is expressed at low levels on the cerebral vas- culature. 3 Therefore, it has been proposed that CD36 is un- likely to be involved in the evolution of cerebral disease. In a recent study on the Kenyan coast, we have shown that disease severity is associated with adhesion of pRBCs to multiple receptors rather than one single receptor (Heddini A and others, unpublished data). In this context, any receptor might, together with the right co-receptors, be of importance in disease severity. We previously described the binding of pRBCs to the en- dothelial receptor PECAM-1/CD31 in vitro. 8 This receptor is a 130-kD glycoprotein belonging to the immunoglobulin superfamily that is expressed on endothelial cells, leuko- cytes, platelets, and some T cell subsets. 12 It is important for maintaining endothelial integrity and also for the process of diapedesis, in which leukocytes traverse the endothelium as they are recruited to a site of inflammation. In both of these cases PECAM-1/CD31 interacts homophilically with other PECAM-1/CD31 molecules. However, heterophilic interac- tions between PECAM-1/CD31 and other structures have also been demonstrated. 13 We have shown that pRBCs ad- here to the N-terminal part of PECAM-1/CD31 and that this binding is mediated by the cysteine-rich inter-domain region (CIDR) and Duffy binding like domain-2 (DBL-2) of P. falciparum erythrocyte membrane protein 1 (PfEMP1). 14 However, it is likely that additional parasite-derived ligands expressed on the surface of pRBCs may also contribute to this binding since treatment of pRBCs with trypsin in con- centrations that will cleave off PfEMP-1 does not completely abrogate PECAM-1/CD31 binding. 15,16 Since the common assays for investigating cytoadherence are dependent on parasitemia to a large extent, we have de- veloped an assay in which soluble fluorescent PECAM-1/ CD31 (sfPECAM-1/CD31) is bound to the surface of pRBCs in suspension. This assay is not dependent on parasitemia and is in good agreement with the adhesion to transfected cell lines. MATERIALS AND METHODS Plasmodium falciparum laboratory strains. In vitro propagated P. falciparum isolates were cultured according to standard procedures 17 with 10% AB + Rh + serum added to the buffered medium (malaria incomplete medium; RPMI 1640 medium supplemented with HEPES (Gibco, Paisley, Scotland), gentamicin (Sigma, St Louis, MO), and sodium bicarbonate (Merck, Darmstadt, Germany). The TM284 strain was isolated in 1990 from a Thai patient with cerebral malaria. TM284 CD31 was obtained by panning TM284 six times on L cells expressing PECAM-1/CD31, and TM284S23 was cloned from a single pRBC bound to PE- CAM-1/CD31 transfected L cells. 15 FCR3S1, a rosette-form- ing clone, was obtained by limiting dilution from the FCR3 strain isolated in the Gambia. Rosette-forming clone FCR3S1.2 and non-rosette-forming clone FCR3S1.6 were obtained from FCR3S1 by single cell cloning. 15,18 FCR3