A Method for Positive and Negative Selection of Plasmodium falciparum Platelet-Mediated Clumping Parasites and Investigation of the Role of CD36

Abstract

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes (IEs) is a frequently observed parasite adhesion phenotype. The importance of clumping in severe malaria and the molecular mechanisms behind this phenomenon are incompletely understood. Three platelet surface molecules have previously been identified as clumping receptors: CD36, globular C1q receptor (gC1qR/HABP1/p32), and P-selectin (CD62P), but the parasite ligands mediating this phenotype are unknown. The aim of this work was to develop a selection method to facilitate investigations of the molecular mechanisms of clumping in selected P. falciparum lines. Magnetic beads coated with anti-platelet antibodies were used to positively and negatively select clumping IEs from P. falciparum strains IT, HB3, 3D7 and Dd2. Clumping in all four positively selected parasite lines was abolished by antibodies to CD36, but was not affected by antibodies to gC1qR or P-selectin. Clumping positive lines showed significantly higher binding to CD36 than clumping negative lines in flow adhesion assays (strains IT, HB3 and 3D7, p<0.05 for all strains, paired t test) and static assays (strain Dd2, p<0.0001 paired t test). However, clumping negative lines IT, HB3 and 3D7 did show some binding to CD36 under flow conditions, indicating that CD36-binding is not sufficient for clumping. These data show that CD36-dependent clumping positive and negative lines can easily be selected from P. falciparum laboratory strains. CD36-binding is necessary but not sufficient for clumping, and the molecular differences between clumping positive and negative parasite lines responsible for the phenotype require further investigation.

Platelet-mediated clumping adhesion phenotypes of P. falciparum-infected erythrocytes: A review

“…The requirement of CD36 for the platelet-mediated clumping phenotype has been reaffirmed in more studies both in field isolates and in laboratory parasite lines (Chotivanich et al, 2004;Wassmer et al, 2008;Mayor et al, 2011;Arman et al, 2013). Platelets with the platelet Naka phenotype that do not express CD36 do not support clumping while anti-CD36 monoclonal antibodies inhibit the clumping phenotype (Pain et al, 2001;Arman et al, 2013) Two more receptors have been reported to mediate platelet-mediated clumping, namely, P-Selectin and gC1qR (Biswas et al, 2007;Wassmer et al, 2008;Mayor et al, 2011).…”

“…Platelet-mediated clumping is a specific adhesion phenotype that is characteristic of a subset of malaria parasites. Previous studies show that the clumping phenotype is sensitive to assay conditions (Arman and Rowe 2008;Arman et al, 2013). The platelet-mediated clumping assay attempts to measure a property of parasites that has not been showed to occur in vivo in clinical malaria, although platelet accumulation in malaria has been reported in the brain of children that died from cerebral malaria (Grau et al, 2003).…”

“…Platelets with the platelet Naka phenotype that do not express CD36 do not support clumping while anti-CD36 monoclonal antibodies inhibit the clumping phenotype (Pain et al, 2001;Arman et al, 2013) Two more receptors have been reported to mediate platelet-mediated clumping, namely, P-Selectin and gC1qR (Biswas et al, 2007;Wassmer et al, 2008;Mayor et al, 2011).…”

“…Knobless IEs are known to Onyambu et al 17 have reduced ability to bind to purified receptors compared to knobbed IEs (Horrocks et al, 2005). Recent published data has shown that clumping parasites can be selected from knobless parasite line DD2 (Arman et al, 2013). It is possible, however, that these parasites are not completely knobless but have a lower expression of the knobby phenotype.…”

The immunological balance between host and parasite in malaria

“…Platelets act as bridging cells between two iRBCs and autoagglutination only occurs with those P. falciparum lines that are able to bind to platelet-CD36, whereas contradictory data exist for globular C1q receptor (gC1qR/HABP1/p32) and P-selectin (CD62P) (Pain et al 2001;Biswas et al 2007;Wassmer et al 2008;Arman et al 2013). Platelet-mediated autoagglutination is associated with CM, since 100% of P. falciparum isolates from Thai patients with CM induced clumping compared to 40%-41% of isolates from uncomplicated or severe non-CM patients (Chotivanich et al 2004).…”