Bimolecular integrinligand interactions quantified using peptide-functionalized dextran-coated microparticles

Integrins play a key role in cellcell and cellmatrix interactions. Artificial surfaces grafted with integrin ligands, mimicking natural interfaces, have been used to study integrin-mediated cell adhesion. Here we report the use of a new chemical engineering technology in combination with single-molecule nanomechanical measurements to quantify peptide binding to integrins. We prepared latex beads with covalently-attached dextran. The beads were then functionalized with the bioactive peptides, cyclic RGDFK (cRGD) and the fibrinogen C-dodecapeptide (H12), corresponding to the active sites for fibrinogen binding to the platelet integrin IIb3. Using optical tweezers-based force spectroscopy to measure non-specific proteinprotein interactions, we found the dextran-coated beads nonreactive towards fibrinogen, thus providing an inert platform for biospecific modifications. Using periodate oxidation followed by reductive amination, we functionalized the bead-attached dextran with either cRGD or H12 and used the peptide-grafted beads to measure single-molecule interactions with the purified IIb3. Bimolecular force spectroscopy revealed that the peptide-functionalized beads were highly and specifically reactive with the immobilized IIb3. Further, the cRGD- and H12-functionalized beads displayed a remarkable interaction profile with a bimodal force distribution up to 90 pN. The cRGDIIb3 interactions had greater binding strength than that of H12IIb3, indicating that they are more stable and resistant mechanically, consistent with the platelet reactivity of RGD-containing ligands. Thus, the results reported here describe the mechanistic characteristics of IIb3ligand interactions, confirming the utility of peptide-functionalized latex beads for the quantitative analysis of molecular recognition. This work focuses on the nanomechanics of bimolecular interactions between the platelet integrin IIb3 and the peptides comprising the integrin-binding sites of natural ligand proteins.