Hl. Goldsmith et al., Time and force dependence of the rupture of glycoprotein IIb-IIIa-fibrinogen bonds between latex spheres, BIOPHYS J, 78(3), 2000, pp. 1195-1206
We studied the shear-induced breakup of doublets of aldehyde/sulfate (A/S)
latex spheres covalently linked with purified:platelet GPIIb-IIIa receptor,
and cross-linked by fibrinogen. Flow cytometry with fluorescein isothiocya
nate-fibrinogen showed than an average of 22,500 molecules of active GPIIb-
IIIa were captured per sphere, with a mean K-d = 56 nM for fibrinogen bindi
ng. The spheres, suspended in buffered 19% Ficoll 400 containing 120 or 240
pM fibrinogen, were subjected to Couette flow in a counter-rotating cone-p
late rheoscope. Doublets, formed by two-body collisions at low shear rate (
G = 8 s(-1)) for less than or equal to 15 min, were subjected to shear stre
ss from 0.6 to 2.9 Nm(-2), their rotations recorded until they broke up or
were lost to view. Although breakup was time dependent, occurring mostly in
the first 2 rotations after the onset of shear, the percentage of doublets
broken up after 10 rotations were almost independent of normal hydrodynami
c force, F-n: at 240 pN, 15.6, 16.0, and 17.0% broke up in the force range
70-150 pN, 150-230 pN, and 230-310 pN, Unexpectedly, at both [fibrinogen],
the initial rate of breakup was highest in the lowest force range, and comp
uter simulation using a stochastic model of breakup was unable to simulate
the time course of breakup. When pre-sheared at low G for >15 min, no doubl
ets broke up within 10 rotations at 70 < F-n < 310 pN; it required >3 min s
hear (>1110 rotations) at F-n = 210 pN for significant breakup to occur. Ot
her published work has shown that binding of fibrinogen to GPIIb-IIIa immob
ilized on plane surfaces exhibits an initial fast reversible process with r
elative low affinity succeeded by transformation of GPIIb-IIIa to a stable
high-affinity complex. We postulate that most doublet breakups observed wit
hin 10 rotations were from a population of young doublets having low number
s of bonds, by dissociation of the initial receptor complex relatively unre
sponsive to force. The remaining, older doublets with GPIIb-IIIa in the hig
h-affinity complex were not broken up in the time or range of forces studie
d.