Time and Force Dependence of the Rupture of Glycoprotein IIb-IIIa-Fibrinogen Bonds between Latex Spheres
| Autor: | Fiona McIntosh, H.L. Goldsmith, Jason Shahin, Mony M. Frojmovic |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Receptor complex
Time Factors Latex Stereochemistry Population Rheoscope Biophysics Platelet Glycoprotein GPIIb-IIIa Complex Dissociation (chemistry) Humans education education.field_of_study Binding Sites Chemistry Fibrinogen Fibrinogen binding Flow Cytometry Microspheres Kinetics Crystallography Covalent bond Stress Mechanical Glycoprotein IIb/IIIa Fluorescein-5-isothiocyanate Protein Binding Research Article |
| Zdroj: | Biophysical Journal. 78(3):1195-1206 |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/s0006-3495(00)76677-x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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