title: Bimolecular integrin-ligand interactions quantified using peptide-functionalized dextran-coated microparticles.
abstract: Integrins play a key role in cell-cell and cell-matrix 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 γC-dodecapeptide (H12), corresponding to the active sites for fibrinogen binding to the platelet integrin αIIbβ3. Using optical tweezers-based force spectroscopy to measure non-specific protein-protein 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 αIIbβ3. Bimolecular force spectroscopy revealed that the peptide-functionalized beads were highly and specifically reactive with the immobilized αIIbβ3. Further, the cRGD- and H12-functionalized beads displayed a remarkable interaction profile with a bimodal force distribution up to 90 pN. The cRGD-αIIbβ3 interactions had greater binding strength than that of H12-αIIbβ3, 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 αIIbβ3-ligand interactions, confirming the utility of peptide-functionalized latex beads for the quantitative analysis of molecular recognition.
authors: Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors: Jessie E P
authors: JE
authors: Sun
authors: Justin
authors: J
authors: Vranic
authors: Russell J
authors: RJ
authors: Composto
authors: Craig
authors: C
authors: Streu
authors: Paul C
authors: PC
authors: Billings
authors: Joel S
authors: JS
authors: Bennett
authors: John W
authors: JW
authors: Weisel
authors: Rustem I
authors: RI
authors: Litvinov
mesh_terms: D001665:Binding Sites; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology*; D003911:Dextrans / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D052898:Optical Tweezers; D010456:Peptides, Cyclic / Q000097:blood; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D003911:Dextrans; D010456:Peptides, Cyclic; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C099250:cyclic (arginyl-glycyl-aspartyl-phenylalanyl-lysyl); D005340:Fibrinogen