Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.