Biomechanics
Single-molecule characterization of the redox regulation of Integrin αIIbβ3—ligand interactions
James Mandeville
Graduate Student
Umass Amherst
Northampton, Massachusetts, United States
Qian Mu, PhD
Graduate Student
Umass Amherst, United States
Yi Wu
Associate Professor
Sol Sherry Thrombosis Research Center, United States
X. Frank Zhang
Professor
University of Massachusetts Amherst, United States
Integrins are heterodimeric cell surface receptors, consisting of both an alpha (α) and a beta (β) subunit. The integrin αIIbβ3 plays an important role in transmembrane signaling as well as thrombosis and hemostasis. It is believed that the protein is regulated through redox reactions involving the 18 cystines that form the 9 disulfide bonds in the αIIb subunit. Of the 9 disulfide bonds, five have been found to be reduced by protein disulfide isomerase and play a direct role in platelet activation. While there is a general idea about how the regulation of these disulfide bonds functions within integrin αIIbβ3, the goal of this research is to understand the cystine redox regulatory mechanism that regulates the mechanical interaction strength between integrin αIIbβ3 and ligands at the single-molecule level.
Atomic force microscopy (AFM) was used to explore force interactions between integrin αIIbβ3 and fibrinogen, a plasma protein and one of the major ligands that interact with integrin αIIbβ3. The integrin was expressed on the surface of human embryonic kidney cells (HEK293) through the co-transfection of plasmids encoding αIIb and β3. Besides the wildtype (WT) αIIb and β3, a mutant αIIb (C515S), which disrupts the disulfide bond at C515 in αIIb and has been shown to enhance integrin-ligand interactions, was used to study the effect of integrin redox regulation. Integrin expression was confirmed by immunofluorescent staining of integrin αIIbβ3 specific antibodies. Human plasma-derived fibrinogen was covalently attached to AFM cantilevers (MLCT-Bio-DC: Bruker Nano), via heterobifunctional PEG linkers. In single-molecule force experiments, the fibrinogen-coated cantilevers were approached and retracted from the cell surface at a predefined speed. If integrin-fibrinogen binding occurs in cantilever-cell contact, the bonded complex(es) will rupture when the cantilever retracts. The unbinding forces between the fibrinogen and both the wild type and mutant integrin ɑIIꞵ3 were measured at various speeds using the force spectroscopy mode of a custom-built AFM. The data was processed using IGOR Pro software to extract the unbinding forces as a function of the loading rate of each unbinding event.
Single-molecule unbinding events were observed between AFM tip-bound fibrinogen and cell surface expressed integrins at a rate of approximately 20%, suggesting the vast majority of the unbinding events stemmed from single integrin-ligand interactions. The Bell-Evans model is applied to extract the force-dependent binding kinetics from the single-molecule data. The plot of the most-probable unbinding force as a function of the loading rate and its fit to the Bell-Evans model is shown in Figure A, which shows that the mutant integrin decreased the unbinding force at lower (< 1000 pN/s) rates but enhanced the unbinding forces at higher loading rates. Figure B shows a prediction of the dissociation rate as a function of pulling force for both the WT and mutant integrin. Significantly, the WT integrin shows lower dissociation rates (i.e., higher affinity) at low forces. However, when force is greater than approximately 50 pN, the affinity of the mutant integrin-ligand interaction becomes higher compared to the WT. Therefore, the comparison between WT and mutant integrins suggests the redox regulatory mechanism at the integrin αIIb subunit could make the integrin more force resistant. Additional experiments are required to further test this hypothesis by testing other disulfides in αIIb under various redox conditions.