Professor Roy R. Hantgan
Department of Biochemistry, Bowman Gray School of Medicine
4 PM, Thursday, Oct. 16, 1997
Room 101, Olin Physical Laboratory
Integrins are widely distributed class of membrane-bound proteins that regulate essential cellular processes ranging from embryonic development to programmed cell death. This talk will focus on the prototypical integrin, the AIIbB3 complex that is present at high density on human blood platelets. Termed the "Responsive Integrin", the AIIbB3 complex binds the adhesive protein fibrin, to the platelet surface as blood clots. Despite major advances in cellular and molecular biology, the structural bases for these adhesive events are not well understood. However, we have obtained some insights into the workings of this "molecular machine" with the tools of biophysical chemistry.
The AIIbB3 complex has been purified from the detergent-solubilized membrane fraction of outdated human platelets by two chromatographic steps: one based on biological function and the next on macromolecular size. Classical (Rayleigh) light scattering yielded a molecular mass of 226 kilodaltons, in agreement with 228 kDa calculated from its amino acid and carbohydrate composition. Dynamic light scattering was used to determine the translational diffusion coefficient of the AIIbB3 complex, as well as the octyl glucoside required for its solubilization. After correction for the contributions of detergent micelles to the intensity autocorrelation function, Dtrans = 3.1+-0.3 x 10-7 cm2 sec-1, corresponding to a Stokes Radius (Rs) of 6.8+-0.7 nm, resulted (Biochemistry, 1993, 32: 3935-3941).
As a spherical, hydrated AIIbB3 complex with bound detergent would exhibit Rs ~5 nm, our data suggested an ellipsoidal particle. Meanwhile, other investigators had presented electron microscopic observations showing the AIIbB3 complex with a globular "head" region ~8 x 12 nm, from which two "tails" ~18 nm in length extended. In collaboration with Dr. Mattia Rocco, Biostructures Unit, IST, Genova, Italy, we examined this issue with hydrodynamic modeling, The AIIb and B3 subunits, as well as a detergent micelle, were represented as a collection of spherical domains. Constraints on the contacts between domains, provided by the biochemical literature, led us to a structural model that resembles the human growth hormone binding protein in its overall geometry. We have since published what may be the "lowest resolution structure" ever presented in Protein Science (1993), 2, 2154-2166.
We are now examining the interactions of the AIIbB3 complex with synthetic peptides corresponding to small fragments of the adhesive protein fibrinogen. Results obtained by light scattering as well as analytical ultracentrifugation indicate these "RGD" peptides promote the reversible oligomerization of the AIIbB3 complex. Since integrin receptors have been shown to form "clusters" on the platelet surface during hemostasis, we are testing the hypothesis that AIIbB3 oligomerization enhances fibrinogen binding by promoting multivalent receptor:ligand links.