INTERACTION FORCES BETWEEN RED-CELLS AGGLUTINATED BY ANTIBODY .4. TIME AND FORCE DEPENDENCE OF BREAK-UP

We report on an extension of a previously described method to measure the hydrodynamic force to separate doublets of fixed, sphered and swollen red cells cross-linked by antibody (S. P. Tha, J. Shuster, and H. L. Goldsmith. 1986. Biophys. J. 50:1117-1126). With a traveling microtube apparatus, doublets are tracked and videotaped in a slowly accelerating Poiseuille flow in 150-mum-diameter tubes, and the hydrodynamic normal force at break-up, F(n), is computed from the measured doublet velocity and radial position. Previous results showed a large range of F(n) the mean of which increased with [antiserum], and an absence of clustering at discrete values of F(n). Since it was assumed that the cells separate the instant a critical force to break all crossbridges was reached, lack of clustering could have been due to the use of a polyclonal antiserum. We therefore studied the effect of monoclonal IgM or IgA antibody on the distribution of F(n). The results showed that the data are as scattered as ever, with F(n) varying from 2 to 200 pN, and exhibit no evidence of clustering. However, the scatter in F(n) could be due to the stochastic nature of intercellular bonds (E. Evans, D. Berk, and A. Leung. 1991 a. Biophys. J. 59:838-848). We therefore studied the force dependence of the time to break-up under constant shear stress (F(n) from 30 to 200 pN), both in Poiseuille and Couette flow, the latter by using a counter-rotating cone and plate rheoscope. When 280 doublets were rapidly accelerated in the traveling microtube and then allowed to coast in steady flow for up to 180 s, 91 % survived into the constant force region; 16% of these broke up after time intervals, t(P), of 2-30 s. Of 340 doublets immediately exposed to constant shear in the rheoscope, 37% broke after time intervals, t(C), from <1 to 10 s. Thus, doublets do indeed break up under a constant shear stress, if given time. The average time to break-up decreased significantly with increasing force, while the fraction of doublets broken up increased. At a given F(n), the fraction of break-ups decreased with increasing [IgM], suggesting that the average number of bonds had also increased. Using a stochastic model of break-up (G. 1. Bell. 1978. Science (Washington DC). 200:618-627; E. Evans, D. Berk, and A. Leung. 1991. Biophys. J. 59:838-848) and a Poisson distribution for the number of bonds, N(b), break-up in slowly accelerating Poiseuille flow and in immediate shear application in Couette flow was simulated. In Poiseuille flow, the observed range and scatter in F(n) could be reproduced assuming [N(b)] > 5. In the rheoscope, the time intervals and number of rotations to break-up, t(C), were quite well reproduced assuming [N(b)] = 4. The similarity of [F(n)] for monoclonal IgM and IgA for doublet break-up under constant slow acceleration is compatible with the conclusion of Evans et al. (1991 a) for normal red cells and Xia et al. (manuscript submitted for publication) for sphered and swollen red cells, that the applied force extracts the antigen from the cell membrane.