Real-time measurements of fibronectin-integrin interactions on polymers using a quartz crystal microbalance with dissipation monitoring (QCM-D)

The hemocompatibility of artificial surfaces is highly dependent on the adhesion and activation of platelets. The integrin GPIIb-IIIa is the major cell surface protein in platelets and functions as receptor for certain adhesive proteins (fibronectin, fibrinogen, von Willebrand factor and vitronectin). So far, this receptor has only been detected on platelets and megacaryocytes. Because surface-adsorbed fibronectin has been shown to support platelet adhesion we investigated the selective adsorption of fibronectin to different biomaterials and the subsequent binding of purified GPIIb-IIIa by real-time measurements using the recently developed QCM-D technique. The QCM-D instrument can measure the adsorbed mass of proteins binding to,I polymer-coated crystal by monitoring the frequency shift in the crystal's resonance frequency and detect changes in the viscoelastic properties of the adsorbed layer by monitoring changes in the energy dissipation. Results show that concentrations of adsorbed fibronectin on test polymers, such as tyrosine-derived poly(DTE carbonate) and poly(lactide glycolide) (PLGA), correlate with the binding capacities for GPIIb-IIIa. Modification of poly(DTE carbonate) by copolymerization with 5% poly(ethylene glycol) (PEG) resulted in decreased surface adsorption of fibronectin followed by reduced binding of GPIIb-IIIa. The binding of GPIIb-IIIa to surface-adsorbed fibronectin was inhibited completely by blocking GPIIb-IIIa with I synthetic inhibitor peptide (GRGDSP). This peptide is based on the cell-attachment sequence (RGD) of fibronectin. Inactive control peptides (GRADSP) did not inhibit the receptor-ligand reaction showing the specificity of the binding reaction. In addition to the changes in the adsorbed mass at the polymer surface, changes in the dissipation may give insights into the structure of the adsorbed protein layer.