Probing the Cytoadherence of Malaria Infected Red Blood Cells under Flow

Abstract

Malaria is one of the most widespread and deadly human parasitic diseases caused by the Plasmodium (P.) species with the P.falciparum being the most deadly. The parasites are capable of invading red blood cells (RBCs) during infection. At the late stage of parasites’ development, the parasites export proteins to the infected RBCs (iRBC) membrane and bind to receptors of surface proteins on the endothelial cells that line microvasculature walls. Resulting adhesion of iRBCs to microvasculature is one of the main sources of most complications during malaria infection. Therefore, it is important to develop a versatile and simple experimental method to quantitatively investigate iRBCs cytoadhesion and binding kinetics. Here, we developed an advanced flow based adhesion assay to demonstrate that iRBC’s adhesion to endothelial CD36 receptor protein coated channels is a bistable process possessing a hysteresis loop. This finding confirms a recently developed model of cell adhesion which we used to fit our experimental data. We measured the contact area of iRBC under shear flow at different stages of infection using Total Internal Reflection Fluorescence (TIRF), and also adhesion receptor and ligand binding kinetics using Atomic Force Microscopy (AFM). With these parameters, we reproduced in our model the experimentally observed changes in adhesion properties of iRBCs accompanying parasite maturation and investigated the main mechanisms responsible for these changes, which are the contact area during the shear flow as well as the rupture area size.

Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

“…Cell flattening has been shown for leukocyte binding (20) and is predicted to affect rolling velocities in adherent parasitized cells (22,23,55), but rolling-velocity measurements with the WSS resolution depicted here have not been previously shown. When pRBCs first bind to CD36 within the microfluidic device the contact patch is likely to be small, but when the WSS increases the cell is pressed against the glass coverslip by the fluid shear forces.…”

“…These unbound pRBCs complicate cell tracking, because unbound cells are not easily differentiated from bound and rolling pRBCs in individual video images. Other cytoadhesion studies have utilized methods to observe both adhesion and detachment rates of cells on substrates (55)(56)(57).…”

“…These WSS-velocity patterns were superficially identical for all parasite clones observed. Atomic force microscopy measurements of CD36 and pRBCs describe a slip-type bond interaction (55,59). Consequently, the change in pRBC-CD36 rolling velocity from 0.8 to 1.3 Pa is unlikely to be due to catch-bond interaction.…”

“…Recent measurements of the pRBC contact patch by total internal reflectance microscopy show that the pRBC contact patch size increases from 0.1 to 0.3 Pa but then remains relatively constant at WSS above 0.5 Pa (55). In that study (55), pRBC rolling velocities were not measured, but taken together, the different approaches are in good agreement that the sudden increase and then leveling out of rolling velocity under increasing WSS may be due to an increase in size of the contact patch under microvascular shear stress, followed by the contact patch size remaining relatively constant and stabilizing the pRBC rolling velocity. During the decreasing WSS experiments, the cell is initially deformed by the high shear stress, so we suspect that the cell contact patch is initially larger and changes more gradually across the measurement.…”

Cytoadhesion of Plasmodium falciparum–infected erythrocytes to chondroitin-4-sulfate is cooperative and shear enhanced

“…[8][9][10][11] Thus, central questions regarding the biomechanical factors that control the dynamics, efficiency, and strength of adhesion are still pending. The literature discusses several factors, including receptor and ligand geometry, bond characteristics, cell deformability, and the area of tight contact between the cell and the surface, that might affect the cytoadhesion of parasitized erythrocytes, [12][13][14][15] but their individual or combined contribution has not yet been experimentally specified.…”