Adhesion of
malaria parasite-infected red blood cells (iRBCs) to microvascular endothelium
is a central event in the pathogenesis of severe falciparum malaria. A biophysical
approach was taken to study the dynamics of this adhesive process using two
different experimental techniques: dual micropipette aspiration and optical
tweezers.
A dual micropipette adhesion assay was used to investigate
the probability and strength of adhesion between an iRBC and an endothelial
cell expressed receptor (CD36) that is important in the pathogenesis of
malaria. CD36 is an important receptor to study, not only is it one of the best
characterized receptor-parasite interactions but it also has an important role
in malaria pathology with respect to its role in severe malaria which is still
not well understood. In fact CD36 blocking intervene has been looked at as treatment strategy for severe malaria, however
little improvement is seen making it vital that we understand more about the
adhesion that occurs between CD36 and iRBCs.
A novel method was employed to assess the adhesion of
individual iRBCs for which the shear elastic modulus was also obtained. This
allowed for the first time the determination of the influence of membrane
rigidity on adhesion. At a constant compression force, an increased membrane stiffness
resulted in a decreased contact area. However, an increased membrane stiffness
resulted in an increased adhesion force, at a constant contact area. An optical
tweezer assay was also employed to measure adhesion. The advantage of this
method is that it provides a means of noninvasive manipulation of objects in
solution, however there are limitations with respect to the maximum force that
can be applied to break the adhesion bonds (~100pN).
The two experimental methods gave comparable results. The adhesion
probability increased with increasing contact time, until approximately 10s
where it remained stable at ~ 40%. A model for 2D kinetic adhesion was fitted
to obtain a kinetic rate of dissociation, kd = 0.11+/-0.02s-1 and kd =
0.089+/-0.025s-1 for the micropipette and optical tweezer method respectively.
The grouped adhesion constant (mrmlKoA) was found to be 0.086 +/- 0.014 using optical
tweezers. Increased contact area resulted in an increase in the adhesion
strength for both methods. The optical tweezermethod further showed an increased
contact time correlated to an increased adhesion strength.
A limitation with these experimental techniques is that they
are inherently non-equilibrium in nature. Consequently, a Langevin simulation
was developed to model the detachment of a bead held in an optical trap, from a
membrane to which it is initially bound to explore the use of fluctuation
theorems to obtain equilibrium values from non-equilibrium work trajectories.
The equilibrium free energy of binding was obtained for various tweezer pulling
rates using fluctuation theorems. Further, umbrella sampling was used to obtain
the equilibrium probability of detachment for a variety of trap potentials.