David F. J. Tees, Ph.D.
Assistant Professor
Ohio University
Using previously developed theory for the hydrodynamic normal, Fn, and shear, Fs, force acting on suspended doublets of sphered, swollen red blood cells (SSRC) and blood group antigen-bearing latex microspheres agglutinated by an antibody to the blood group B antigen (Tha et al., Biophys. J. 50:1117, 1986), the effect of force on the time dependence of break-up was studied. Forces were applied using either a travelling microtube in which doublets were exposed to slowly accelerating Poiseuille flow in 150 mm diameter tubes, or in a cone and plate Rheoscope which allowed virtually instantaneous application of Couette flow. Break-up was observed while tracking individual doublets under the microscope. Originally, the force was increased at a steady rate, and it was assumed that the doublets separate the instant a critical force to "break all crossbridges" was reached. This force per bond should be manifest in a distribution of Fn as equally spaced peaks corresponding to one, two, three, etc. bonds. It was first postulated that the lack of such clustering was due to the use of a polyclonal antiserum as the agglutinating agent. We therefore studied the effect of monoclonal IgM antibody on the distribution of Fn. The results showed that the data is as scattered as ever, with Fn at break-up varying from 2 to 200 pN. The continued lack of clustering at discrete values of Fn suggested that the scatter was due to the stochastic nature of intercellular bonds (Evans et al., Biophys. J. 59:838, 1991), a result confirmed by using the Rheoscope to apply a virtually instantaneous constant shear rate and observing break-up at a distribution of times under a constant maximum Fn (from 30-200 pN). With increasing force the fraction of doublets broken up increased. At a given Fn, the fraction of doublets broken up decreased with increasing [IgM], suggesting that the average number of bonds had increased. Using a stochastic model of break-up (Bell, Science 200:618, 1978) and a Poisson distribution for the number of bonds, Nb, break-up in Poiseuille and Couette flow was simulated. In Poiseuille flow, the observed range and scatter in Fn could be reproduced assuming <Nb> > 5. In the Rheoscope, fraction of doublets broken up and the time distribution of break-up were quite well reproduced assuming <Nb> = 4.
Studies using micropipette aspiration (Evans et al., Biophys. J. 59:838, 1991) and shear flow (Xia et al. Biophys. J. 66:1222, 1994) indicate that bond rupture can occur through extraction of antigen from the cell membranes of SSRC. Accordingly, the Rheoscope experiments were repeated using 4.62 um carboxyl modified latex microspheres with covalently coupled synthetic blood group B trisaccharide antigen. Comparison of the time and force dependence of break-up showed that antigen spheres required significantly greater forces to achieve the same degree of doublet break-up as SSRC. The computer simulation showed that the reduced sensitivity to force is due to a change in bond character (the range and depth of the bond energy minimum) and not to an increase in the number of bonds linking antigen sphere doublets. This is consistent with the anticipated increased force required to break antibody-antigen bonds compared with that required to extract a receptor from the membrane, although changes of receptor substrate from cell to latex and the possibility of latex strand extraction from the microspheres are possible complicating factors.