Time and force dependence of the rupture of glycoprotein IIb-IIIa-fibrinogen bonds between latex spheres.
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 isothiocyanate-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 binding. The spheres, suspended in buffered 19% Ficoll 400 containing 120 or 240 pM fibrinogen, were subjected to Couette flow in a counter-rotating cone-plate rheoscope. Doublets, formed by two-body collisions at low shear rate (G = 8 s(-1)) for < or =15 min, were subjected to shear stress 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 hydrodynamic 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 computer 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 doublets broke up within 10 rotations at 70 < F(n) < 310 pN; it required >3 min shear (>1110 rotations) at F(n) = 210 pN for significant breakup to occur. Other published work has shown that binding of fibrinogen to GPIIb-IIIa immobilized on plane surfaces exhibits an initial fast reversible process with relative low affinity succeeded by transformation of GPIIb-IIIa to a stable high-affinity complex. We postulate that most doublet breakups observed within 10 rotations were from a population of young doublets having low numbers of bonds, by dissociation of the initial receptor complex relatively unresponsive to force. The remaining, older doublets with GPIIb-IIIa in the high-affinity complex were not broken up in the time or range of forces studied.
Goldsmith
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HL
McGill University Medical Clinic, Montreal General Hospital Research Institute, Montreal, Quebec, Canada. mohg@musica.mcgill.ca
McIntosh
F A
FA
Shahin
J
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Frojmovic
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MM
D001665:Binding Sites; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007700:Kinetics; D007840:Latex; D008863:Microspheres; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013314:Stress, Mechanical; D013997:Time Factors
D007840:Latex; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate